The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Isoprenoids, such as squalene, are commercially important types of lipids. They have excellent lubricity, oxidative stability, low pour points, low freezing points, high flash points, and facile biodegradability. Squalene is currently produced by extraction from olive oil or cold water shark liver oil at a high unit cost. Because of the high unit cost, economically feasible uses for squalene and squalene (the fully hydrogenated derivative of squalene) are in small market applications such as watch lubricants, pharmaceutically/nutraceuticals, cosmetics, perfumes and as chemical intermediates for high-value products.
There exist, however, significant potential markets for biodegradable lubricants, lubricant additives, and hydraulic fluids. Biodegradability of these products is particularly important for environmentally sensitive applications, such as agricultural applications, or where considerable lubricant or hydraulic fluids may be lost to the environment. The potential markets for biodegradable lubricants, lubricant additives, and hydraulic fluids are quite large, estimated to be on the order of five million metric tons per annum.
Biodegradable lubricants, lubricant additives, and hydraulic fluids derived from vegetable and animal fats and oils are available, but they have drawbacks. They typically solidity at relatively high temperatures (i.e., they solidity in cold weather) and have flash points that are too low for use in hot conditions, (i.e., they break down or combust under normal hot engine conditions).
Thus, a cost effective method of production of squalene is desired that would allow for large-scale manufacturing and widespread use of squalene and squalane in biodegradable lubricants, lubricant additives, and hydraulic fluids.
Chang et al., (Appl. Microbiol. Biotechnol., 2008, 78, 963-72) discloses the discovery of a wild type yeast, Pseudozyma sp. JCC207, that produces “a large amount of squalene and several polyunsaturated fatty acids.” Chang et al. describe isolating Pseudozyma sp. JCC207 from seawater collected near Guam, USA, and are unsure whether Pseudozyma sp. JCC207 is a new species or a variant of P. regulosa or P. aphidis. In the article, “the efficiency of squalene production [of Pseudozyma sp. JCC207] was investigated under different conditions.”
Dow AgroSciences LLC, Using Yeast Fermentatin to Produce Cost-Effective and Biodegradable Lubricants, http://statusreports.atp.nist.gov/reports/95-01-0148PDF.pdt, discloses that “[t]he company proposed to use genetic engineering to alter the metabolic characteristics of an oleaginous (oily) yeast to increase the yeast's ability to produce isoprenes through biosysnthesis.” Specifically, four enzymes were targeted: ACCase, hydroxymethylglutaryl CoA reductase (HNGR), squalene synthetase, and squalene epoxidase.
U.S. Pat. No. 5,460,949 discloses “[a] method increasing the accumulation of squalene and specific sterols in yeasts.” In particular, it is disclosed that “[s]qualene and sterol accumulation is increased by increasing the expression level of a gene encoding a polypeptide having the HMG-CoA reductase activity.”